The partial pressure of oxygen in a solid tumor is not uniform, and tumor cells are exposed to various oxygen environments. This results from the fact that the scattering distance of oxygen molecules from a blood vessel to the tissue of a tumor is limited. In the field of radiobiology, the inside of a solid tumor is divided into three regions, that is, 1) an aerobic region, 2) a dead region and 3) an hypoxic region according to the amount of oxygen supplied to each tumor cell.
1) Since an extremely large number of oxygen molecules are supplied to each tumor cell at a distance from a capillary vessel of up to 70 μm, this region is called “an aerobic region”. Oxygen molecules are indispensable for the acquisition of the effect of radiotherapy for solid tumors. Therefore, it is known that the aerobic region rich in oxygen molecules literally is a region having extremely high radiation sensitivity. The treatment effect of chemotherapy for the aerobic region is considered to be high because an anti-cancer drug is easily scattered from a blood vessel system to the aerobic region when chemotherapy is performed.
2) Oxygen molecules released from the blood vessel are consumed by tumor cells in the aerobic region while the molecules are scattered. Therefore, oxygen molecules required for survival of tumor cells existent in a region far from the capillary vessel are not supplied to the cells. As a result, tumor cells far from the blood vessel system at a distance of 70 μm or more die, thereby forming a dead region.
3) A hypoxic region composed of hypoxic cells is existent between the aerobic region and the dead region. The minimum amount of oxygen molecules required for the survival of tumor cells is supplied to the hypoxic region. However, oxygen molecules enough to obtain the effect of radiotherapy are not supplied. Therefore, the hypoxic region in the tumor has extremely low radiation sensitivity, which is considered to be one of the causes of the re-proliferation of tumor cells after the end of radiotherapy. Since the amount of an anti-cancer drug scattered from the blood vessel system to the hypoxic region is limited when a chemical treatment is performed, a satisfactory treatment effect cannot be expected in fact.
It has been difficult to confirm the existence of such a hypoxic cell. This is because there has been substantially no means of monitoring the existence of oxygen in a cell. Known as means of monitoring the hypoxic cells are a method of measuring an oxygen voltage of a cell using micro-electrodes, immunocytostaining using Pimonidazole (Hypoxyprobe-1) known as a hypoxic cell indicator, immunocytostaining using a gene product whose expression is induced in a cell under hypoxic conditions as a label, and the like. However, the above methods are technically difficult, and at present, also apparatuses for carrying out these are complex and not generally used. That is, the development of general-purpose means of detecting hypoxic cells has been desired.
As described above, in cancer treatment, the existence of hypoxic cancer cells hinders the treatment by radiotherapy or chemotherapy drug. Means of removing these cells effectively has been desired. Although only the use of a combination of a hypoxic cell radiation sensitizer and radiotherapy has been known to cope with the hypoxic cells, only one drug is now in a clinical trial stage and there does not currently exist a hypoxic cell radiation sensitizer which has been put to practical use. This results from the fact that 2-nitroimidazole which is the mother nucleus of the hypoxic cell radiation sensitizer is neurotoxic and it is difficult to control its toxicity and medical effect. That is, the development of means of getting rid of a hypoxic cancer cell effectively has been desired.
By the way, the expression of physiologically important genes such as a vascular endothelial growth factor (VEGF) and erythropoietin (EPO) is induced in a cell under a hypoxic environment. The expression of these genes is induced by hypoxia-inducible factor-1 complex (hereinafter, referred to as HIF-1) in a transfer level.
HIF-1 is a heterodimer consisting of HIF-1α protein and HIF-1β protein. These sub-units each have a domain for binding to DNA called “basic-helix-loop-helix domain (bHLH domain)” and a domain for forming a heterodimer called “PER-aryl hydrocarbon nuclear translocator (ARNT)-SIM (PAS) domain” at N termini. It has been found that the HIF-1α protein has two transactivation domains, that is, N- and C-transactivation domains (N-TAD, C-TAD).
The activation mechanism dependent on the oxygen concentration of HIF-1 has recently been clarified. The transfer and translation of HIF-1β mRNA are always activated and its gene product (protein) is always expressed non-dependent on the partial pressure of oxygen in the outside world. Although the transfer and translation of HIF-1α mRNA are also always activated, the biosynthesized HIF-1α protein is positively degraded under aerobic conditions and is existent stably only under hypoxic conditions.
It has thus been found that the stability of the HIF-1α protein is controlled dependent on the concentration of oxygen in the outside world and that the transfer activity of HIF-1 is controlled dependent mainly on the amount of the protein.
To date, it has been reported that the 401a.a.-603a.a. domain is important for the stabilization of HIF-1α under hypoxic conditions in experiments using a partially deleted mutant of HIF-1α (Huang L E, Gu J, Schau M and Bunn H F. 1998. Regulation of hypoxia-inducible factor 1 alpha is mediated by an O2-dependent degradation domain via. the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA. 95:7987-7992: document 1). This domain is called “an Oxygen Dependent Degradation domain” (ODD domain).
It has been known that the oxygen dependent stability of HIF-1α suggests that amino acid residues in this domain be modified dependent on oxygen under aerobic conditions, be ubiquitinated in the end, and be degraded by proteasome (Huang, L. E., Gu, J. Schau, M. and Bunn, H. F. Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA. 95: 7987-7992, 1998).
Therefore, it has also been known that HIF-1α can obtain the same stability as that under hypoxic conditions by culturing HIF-1α in a medium containing a proteasome inhibitor such as N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (to be abbreviated as “Cbz-LLL” hereinafter) (Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and Goldberg, A. L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761-771, 1994: document 3). And this is disclosed (Sutter, C. H., Laughner, E. and Semenza, G. L. Hypoxia-inducible factor 1α protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations. Proc. Natl. Acad. Sci. USA. 97: 4748-4753, 2000: document 4). Through this fact, it has been assumed that the degradation of a fused protein containing the polypeptide is carried out through a degradation mechanism by ubiquitin-proteasome like HIF-1α and that the fused protein is stabilized under hypoxic conditions and in a medium containing Cbz-LLL.
It has been reported that the Gal-4 protein fused with 530a.a.-652a.a. of the HIF-1α protein is controlled to be positively degraded in a cultured cell only when the concentration of oxygen is high (Vickram Srinivas, Li-Ping Zhang, Xiao-Hong Zhu and Jaime Caro. 1999. Characterization of an Oxygen/Redox-Dependent Degradation Domain of Hypoxia-Inducible Factora (HIFα) Proteins. Biochem. Biophy. Res. Com. 260: 557-561: document 5). It has also been reported that when HIF-1α561a.a.-568a.a. in a gene fused with Gal-4 and 529a.a-826a.a. of HIF-1α is substituted by the alanine residue, the above control is lost. It is presumed, from this fact, that the domain around 561a.a.-568a.a. of HIF-1α where the HIF-1α of a mouse and human HIF-1α are well kept takes part in the oxygen concentration dependent stabilization of a protein. It is also discussed whether the 557a.a.-571a.a. domain of HIF-1α plays an important role in the control of the stability of the HIF-1α protein (above document 5).
However, the inventors of the present invention have found it impossible to make the stabilization of a fused protein dependent on the concentration of oxygen only with the 557a.a.-571a.a. domain. It cannot be said that the domain taking part in the stabilization of a fused protein of the HIF-1α protein is identified.
It was reported in 1988 that a protein called “TAT” derived from a human immunodeficiency virus (HIV) has the activity of transducing a protein through cell membrane (Cell; 55, 1179 (1988), Proc. Natl. Acad. Sci. USA; 91, 664 (1994)). After that, it was elucidated that a domain consisting of only 11 amino acids of TAT protein (TAT protein transduction domain) has the above activity. At the same time, it was also reported that β-galactosidase protein fused with this TAT protein transduction domain is introduced into a cell.
However, the relationship between the above HIF-1α and TAT and the relationship between HIF-1α and a protein having protein transduction activity through membrane have been unknown so far. Therefore, it has been unknown that when the HIF-1α protein having a specific region for controlling the stabilization of the HIF-1α protein, a protein having protein transduction activity through membrane and other protein are fused together, making use of the specific region, the obtained fused protein can be introduced into a cell and that in the cell harboring the fused protein, oxygen-dependent stability can be imparted to the fused protein.